KSHV is the causative agent of Kaposi's sarcoma (KS) that occurs most frequently and severely in HIV-AIDS. KSHV has also been implicated in several lymphoid disorders, including pleural effusion lymphoma (PEL) and Castleman's disease. Long-term latent infection in B-lymphocytes and persistent infection in endothelial cells is thought to be a major driving force for KSHV-associated pathogenesis. Latent infection is characterized by the transcription of several viral genes associated with viral genome stability and host-cell survival. The major latency transcript is a multicistronic message that consists of the LANA, vCyclin, and vFLIP genes, which can be alternatively spliced to generate the viral microRNAs (vmiRNAs) and Kaposin genes. The latency transcriptional control is also responsible for the regulation of the vGPCR gene, which has been implicated in KS pathogenesis. Proper regulation of these viral transcripts is critical for viral genome persistence during latency and for pathogenesis in KSHV infected lesions. Our preliminary data indicates that this region of the viral chromosome is protected from epigenetic silencing by the chromatin insulator protein CTCF and colocalization with cohesin subunits (e.g. SMC1, SMC3, Rad21). In addition, changes in CTCF-cohesin interactions contribute to the cell cycle activation of vGPCR in latently infected PEL cells. We have also found that these CTCF sites in the latency control region are important for the programming of RNA polymerase II elongation and mRNA processing.
In Aim 1, we will determine how the CTCF-cohesin complex contributes to the regulation of latency transcription, and protects the major latency control region from epigenetic silencing. We have also found that viral encoded miRNAs contribute to the epigenetic programming of the viral chromosome during latency. We propose in Aim 2 of this proposal to determine whether CTCF-cohesin complex regulates vmiRNA expression, and additionally, whether vmiRNAs coregulate the function of CTCF- cohesins in regulating the epigenetic state of the latent viral chromosome. Finally, we propose to investigate the mechanism of a small molecule inhibitor that selectively deregulates KSHV latency transcription. Glychyrrizic acid (GA) is a bioactive natural product derived from licorice that selectively inhibits KSHV positive PEL cell proliferation. We have found that GA deregulates RNA polymerase II interactions with the CTCF- cohesin complex at the LANA 5'UTR. We have used chemical affinity to identify candidate target molecules of GA.
In Aim 3, we propose to further characterize the mechanism of GA deregulation of LANA transcription and its potential targeting of the CTCF-cohesin complex. Together, these aims focus on a novel mechanism of gene regulation that maintains latency gene expression during latency, restricts the pathogenic expression of vGPCR, and may serve as a potential target for small molecule intervention in KSHV-associated disease.

Public Health Relevance

Human herpesvirus 8 (HHV8), more commonly referred to as Kaposi's Sarcoma (KS)-Associated Herpesvirus (KSHV) is the etiological agent of HIV-associated and endemic forms of KS. KSHV has also been linked to two other lymphoproliferative disorders, primary effusion lymphoma (PELs) and multicentric Castleman's disease. Additional diseases may also be associated with KSHV, including autoimmune disorders like multiple sclerosis and system lupus erythematosis. In all cases of KSHV, the infection is a complex mixture of latent episomes and lytic productive infection. In this application, we investigate the mechanisms regulating gene expression from KSHV latent genomes, and how small molecule inhibitors may be developed to disrupt this process. Further elucidation of the molecular mechanisms regulating latency transcription will provide useful information for understanding and treating viral pathogenesis and disease.